Fig 2: DPSC-derived neurons from PWS patients have impaired endosome-mediated protein trafficking and decreased SG protein abundance.(A) Representative images of immunofluorescence staining of M6PR in DPSC-derived neurons. Proper M6PR trafficking shows clustered juxtanuclear localization. (B) Proportion of cells with impaired M6PR trafficking is significantly increased in PWS and SYS DPSC-derived neurons. Each data point represents 1 individual, plotted as mean ± SD; more than 75 cells/data point, analyzed by 1-way ANOVA. (C) Representative images of immunofluorescence staining of integrin a5 in DPSC-derived neurons. Yellow arrows indicate increased intracellular (endosomal) pools. (D) Quantification of Western blot analysis showed reduced expression of integrin a5, aV, and ß1 in PWS deletion DPSC-derived neurons. Each target protein is first normalized to GAPDH, then normalized to averaged control. Each data point represents 1 individual (n = 6), plotted as mean ± SD and analyzed by unpaired, 2-tailed t test. (E) Representative images of immunofluorescence staining of phalloidin on VPS35-marked endosomes in DPSC-derived neurons. (F) Fluorescence intensity of F-actin on VPS35-marked endosomes is significantly decreased in PWS and SYS DPSC-derived neurons. Each data point represents 1 individual, plotted as mean ± SD; more than 75 cells/data point, analyzed by 1-way ANOVA. (G) Fluorescence intensity of ArpC5 on VPS35-marked endosomes is significantly decreased in PWS and SYS DPSC-derived neurons. Each data point represents 1 individual, plotted as mean ± SD; more than 75 cells/data point, analyzed by 1-way ANOVA. (H) Western blot analysis of M6PR, a5, aV, and ß5 between control and PWS iN. GAPDH served as loading control. (I) Quantification of Western blot analysis showed reduced expression of PCSK1, PCSK2, and CHGB in PWS deletion and SYS DPSC-derived neurons. Each target protein is first normalized to GAPDH, then normalized to averaged control. Each data point represents 1 individual, plotted as mean ± SD and analyzed by unpaired, 2-tailed t test. *P < 0.05 and **P < 0.01. Scale bars: 10 µm.

Fig 3: Differential role of ARPC5 isoforms in replication stress-mediated nuclear F-actin (NFA) formation.(A) Schematic of experimental setup showing timeline of knockout (KO) generation and induction of replication stress in the JNLA KO cells using aphidicolin (APH). See related Figure 6—figure supplement 1A. (B) Shown are representative spinning disk confocal still images (maximum projection) of the APH pretreated KO or control cells. The movies were acquired for 5 hr with acquisition every 15 min post pretreatment of cells with APH. The stills at the indicated time points are representative of the time point where the maximal NFA burst has been observed in each condition. (C) Stacked bar graph (denoted by three different colors for each condition) shows the kinetics of NFA burst throughout 5 hr of live-cell imaging duration, with maximum NFA burst observed within the first 2 hr. (D) Quantification of the % of cells with NFA bursts (within the first 2 hr of imaging) post replication stress induction in control and KO cells. Each data point indicates the mean value of an independent experiments with at 40–60 analyzed per condition with indicated mean ± SD from three independent experiments. Statistical significance was calculated using one-way ANOVA (Kruskal–Wallis test). Also see related Figure 6—figure supplement 1C for single-cell tracks. (E) JNLA cells transduced with either mCherry (control) or CAMBP4.NLS-mCherry were pretreated with solvent control (DMSO) and were either activated by PMA + ionomycin (P/I) or treated with APH for induction of replication stress for 3 hr prior to live-cell imaging. Shown are maximum projection of representative spinning-disk confocal still images (showing the time frame where maximum NFA burst was observed) in the DMSO control compared with either P/I or APH-mediated NFA bursts (white arrows) in the presence and absence of nuclear calmodulin. Movies for visualizing replication stress were acquired for 5 hr with acquisition every 15 min post pretreatment of cells. Whereas movies for visualizing P/I activation-induced NFA were acquired for 5 min with acquisition every 15–30 s. Also see related Figure 6—figure supplement 1E and F. (F) Quantification of NFA in the abovementioned conditions was performed; each data point indicates the mean value of an independent experiment with at least 30 cells analyzed per condition with indicated mean ± SD from three independent experiments. Statistical significance was calculated using Welch’s t-test. *p=0.0332, **p=0.0021, ***p=0.0002, and ns: not significant. Scale bar, 3 µm. Also see related Figure 6—figure supplement 1B.

Fig 4: Mutant ARPC5 biochemical studies.a Western blotting analysis of patient 1 (P1), her parents, and healthy controls (HC) T-cell blasts lysates with antibodies specific to Arp2/3 complex subunits, as indicated. Vinculin and ß-tubulin were used as loading controls. b Western blotting analysis of cell extracts from P1’s and HC’s fibroblasts after native gel electrophoresis. ARPC5- or ARPC2-specific antibodies were used to probe the Arp2/3 complex; GAPDH was used as a loading control. Blots in this figure are representative of at least two independent experiments. The numbers below the western blotting images (a and b) represent protein expression levels, quantitatively measured in relation to healthy controls, after normalization to the loading control. Healthy controls’ average value was set at 10. PAGE polyacrylamide gel electrophoresis. Source data are provided as a Source Data file.

Fig 5: Effects observed on nuclear and plasma membrane F-actin dynamics upon ARPC5/C5L knockout and its impact on proximal TCR signaling and cytokine production.(A) Shown are representative maximum intensity projections of confocal still images of the indicated KO JNLA cells overexpressing mCherry (control) or mCherry fusion proteins of the respective ARPC5 isoforms, post activation with either anti-CD3+CD28 antibodies (top panel) or with PMA/ionomycin (P/I) (bottom panel). Arrows point to the nuclear F-actin (NFA, bottom). Scale bar, 3 µm. Arrowheads point to the F-actin ring (top). Scale bar, 5 µm. See related Figure 4—figure supplement 1A-D and Figure 4—figure supplement 2A for western blots of KO and overexpression and Figure 4—figure supplement 2B and D for NFA data. (B) Quantification of F-actin ring (AR) formation in the PM, stained with Phalloidin-647, is compared to the NFA formation visualized with NLA-GFP in the respective KO or KO+ARPC5 isoform expressing cells was performed relative to the non-targeting control (NTC)-treated cells. ‘mCherry’ alone was used as vector backbone control for the overexpression study. Bars indicate mean from three independent experiment where 30–40 cells was analyzed per condition. One-way ANOVA with Kruskal–Wallis test was used to determine statistical significances, where **p=0.0021 and ns: not significant. See related Figure 4—figure supplement 2C,E. (C, F) Representative confocal images of JNLA.GFP cells with indicated knockout or control (NTC) upon 5 min of activation on coverslips coated with anti-CD3+CD28 antibodies. Cells were fixed and stained for F-actin (with Phalloidin 488) and pTyr or pSLP-76 (Alexa Fluor 647), respectively. (D and G) show quantification of the total number of pTyr or pSLP-76 clusters/cell in KO condition relative to control cells analyzed using the ‘Spot Detector’ Fiji plugin. Data presented here are mean ± SD from three independent experiments in which two technical replicates were measured per sample. (E, H) Dot plots represent the changes in overall intensity of pTyr or pSLP-76 clusters per cell where each dot represents intensity of clusters/cell analyzed manually using Fiji. In (B, D, E), each data point indicates the mean value of an independent experiments with at least 80 cells analyzed per condition with the indicated mean ± SD from three independent experiments. One-sample t-test was used to determine statistical significances, where *p=0.033, **p=0.0021, ***p=0.0002 and ns: not significant. Scale bar, 5 µm. (I, J) Cytokine production of A30.1 cells treated with the indicated shRNAs in response to P/I. Bars show intracellular levels of TNFa or IL-2, 4 hr or 16 hr of post activation relative to cells treated with the scrambled shRNA control. One-way ANOVA with Tukey’s multiple-comparison test was used to determine statistical significances ( **p=0.005, ***p=0.0002, ****p=0.0001, and ns: not significant). Relates to Figure 4—figure supplement 3A–E.